Initial data decontamination using species occupancy detection modeling on a per-sample, per-locus basis. This is based on Ryan Kelly’s work/GitHub tutorial.
The input is a table of counts per sample/locus that only includes the ASVs with taxonomic hits in the BLAST database (since we wouldn’t be drawing inference from the sequences without taxonomic info).
The input dataframe is ../data/feature_df_no_eDNA_metazoa_only.rds and is generated in 01-updated-background-Oct-2020.Rmd.
Mostly, we are interested in the vouchered reference DNA pool, since the full reference DNA pool and experimental feed mixtures almost certainly included contamination from samples obtained from seafood markets.
Most of the action is now wrapped up in a function called sodm.by.locus which outputs files to “csv_outputs/” in the current working directory.
# function source
source("../R/metabarcoding-funcs.R")
library(tidyverse)
library(stringi)
library(here)
here() starts at /Users/dianabaetscher/Documents/git-repos/metabarcoding-primer-portfolio
library(rstan)
Loading required package: StanHeaders
rstan (Version 2.19.3, GitRev: 2e1f913d3ca3)
For execution on a local, multicore CPU with excess RAM we recommend calling
options(mc.cores = parallel::detectCores()).
To avoid recompilation of unchanged Stan programs, we recommend calling
rstan_options(auto_write = TRUE)
Attaching package: ‘rstan’
The following object is masked from ‘package:tidyr’:
extract
library(shinystan)
Loading required package: shiny
Registered S3 method overwritten by 'htmlwidgets':
method from
print.htmlwidget tools:rstudio
This is shinystan version 2.5.0
library(bayesplot)
This is bayesplot version 1.7.1
- Online documentation and vignettes at mc-stan.org/bayesplot
- bayesplot theme set to bayesplot::theme_default()
* Does _not_ affect other ggplot2 plots
* See ?bayesplot_theme_set for details on theme setting
library(broom)
options(mc.cores = parallel::detectCores())
rstan_options(auto_write = TRUE)
set.seed(108)
setwd(here())
The working directory was changed to /Users/dianabaetscher/Documents/git-repos/metabarcoding-primer-portfolio inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.
knitr::opts_chunk$set(echo = TRUE)
Two Stan model functions for two steps in this process:
From Ryan Kelly’s github page:
Stan actually does its work in C++, so it has to write out a program to a file (outside of R) and compile it to run at a deeper level of your computer’s architecture. This takes a little while the first time you compile a given set of Stan code, but only needs to be redone if you change the Stan code.
##Stan Model
modelText <- "data{/////////////////////////////////////////////////////////////////////
int<lower=1> S; // number of sites or samples (nrow)
int<lower=1> K[S]; // number of replicates per site (ncol)
int<lower=0> N[S]; // detected or not at that site/replicate
int z[S];
}
parameters{/////////////////////////////////////////////////////////////////////
real<lower=0,upper=1> psi; //commonness parameter
real<lower=0,upper=1> p11; //true positive detection rate
real<lower=0,upper=1> p10; //false positive detection rate
}
transformed parameters{/////////////////////////////////////////////////////////////////////
}
model{/////////////////////////////////////////////////////////////////////
real p[S];
for (i in 1:S){
z[i] ~ bernoulli(psi);
p[i] = z[i]*p11 + (1-z[i])*p10;
N[i] ~ binomial(K[i], p[i]);
};
//priors
psi ~ beta(5,5);
p11 ~ beta(5,5);
p10 ~ beta(1,50);
}
generated quantities{
real<lower=0,upper=1> Occupancy_prob[S]; //after inferring parameters above, now calculate occupancy probability for each observation. Equation from Lahoz-Monfort et al. 2015
for (i in 1:S){
Occupancy_prob[i] = (psi*(p11^N[i])*(1-p11)^(K[i]-N[i]))
/ ((psi*(p11^N[i])*(1-p11)^(K[i]-N[i]))
+ (((1-psi)*(p10^N[i]))*((1-p10)^(K[i]-N[i])))
);
}
}
"
write.table(modelText, "Stan_SOM_demo.stan", row.names = F, quote = F, col.names = F)
And we can then explore the posterior occupancy probability for all possible outcomes of 10 trials, given these parameters:
modelText <- "data{/////////////////////////////////////////////////////////////////////
int<lower=1> S; // number of sites or samples (nrow)
int<lower=1> K[S]; // number of replicates per site (ncol)
int<lower=0> N[S]; // detected or not at that site/replicate
int z[S];
}
parameters{/////////////////////////////////////////////////////////////////////
real<lower=0,upper=1> psi; //commonness parameter
real<lower=0,upper=1> p11; //true positive detection rate
real<lower=0,upper=1> p10; //false positive detection rate
}
transformed parameters{/////////////////////////////////////////////////////////////////////
}
model{/////////////////////////////////////////////////////////////////////
real p[S];
for (i in 1:S){
z[i] ~ bernoulli(psi);
p[i] = z[i]*p11 + (1-z[i])*p10;
N[i] ~ binomial(K[i], p[i]);
};
//priors
psi ~ beta(5,5);
p11 ~ beta(5,5);
p10 ~ beta(1,50);
}
generated quantities{
real<lower=0,upper=1> Occupancy_prob[11]; //after inferring parameters above, now calculate occupancy probability for each observation. Equation from Lahoz-Monfort et al. 2015
for (i in 0:10){
Occupancy_prob[i+1] = (psi*(p11^i)*(1-p11)^(10-i))
/ ((psi*(p11^i)*(1-p11)^(10-i))
+ (((1-psi)*(p10^i))*((1-p10)^(10-i)))
);
}
}
"
write.table(modelText, "Stan_SOM_allPossibilities.stan", row.names = F, quote = F, col.names = F)
The dataframe that just includes ASVs associated with taxonomy retained
features_w_taxonomy <- readRDS("../extdata/downsampled_loci/data/feature_df_no_eDNA_metazoa_only.rds")
Cycle over a list of the loci for a given site.
loc_list
[1] "16SH1" "16Svar" "18SSSU3" "18Sn4" "L2513H2714" "aquaF2"
[7] "aquaF3" "cep" "ceph16S" "crust16S" "fishcoilbc" "fishminiA"
[13] "mifish" "minibar" "nsCOIFo" "plankCOI" "shark474" "sharkCOImini"
[19] "short28S" "crust2" "16Sfish" "teleo"
Cycle over the loci and generate csv outputs that I can then filter based on occupancy probabilties.
# lapply over the list of 22 loci
# the site, dataframe, and number of replicates are consistent for now
lapply(loc_list, sodm.by.locus, feature_table = features_w_taxonomy, site = "VRP", n_replicates = 9)
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Error in data.frame(K = n_replicates, N = rowSums(wide.loc.matrix), z = ifelse(rowSums(wide.loc.matrix) > :
arguments imply differing number of rows: 1, 0
loc_list
[1] "16SH1" "16Svar" "18SSSU3" "18Sn4" "L2513H2714" "aquaF2"
[7] "aquaF3" "cep" "ceph16S" "crust16S" "fishcoilbc" "fishminiA"
[13] "mifish" "minibar" "nsCOIFo" "plankCOI" "shark474" "sharkCOImini"
[19] "short28S" "crust2" "16Sfish" "teleo"
Because we only really could control for false positives (to some degree) in the vouchered reference pool, my expectation is that those samples will have greater separation between true and false positives, and thus, perform better using this framework.
I have always seen an output error message from this analysis that says: Tail Effective Samples Size (ESS) is too low, indicating posterior variances and tail quantiles may be unreliable. Running the chains for more iterations may help. See http://mc-stan.org/misc/warnings.html#tail-ess
Although I bumped up the number of iterations from 5K to 10K, I never fully investigated if I could get the warning message to go away with a sufficient number of iterations.
Before I move forward with these data, let’s see if 50K or 100K improves the estimate or if it is a sample size problem that I’m not going to be able to overcome by more iterations.
Based on testing a few different iteration options, I’m sticking with 100k.
Given the uncertainty of the tail probabilities, it seems best to take that into consideration when removing ASVs as putative false positives.
So, if the occupancy estimate + the error is <80%, I will say that is sufficient evidence to remove that ASV.
Full reference pool - all 22 loci
# lapply over the list of 22 loci
lapply(loc_list, sodm.by.locus, feature_table = features_w_taxonomy, site = "FRP", n_replicates = 9)
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Tail Effective Samples Size (ESS) is too low, indicating posterior variances and tail quantiles may be unreliable.
Running the chains for more iterations may help. See
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Chain 1:
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SAMPLING FOR MODEL 'Stan_SOM_allPossibilities' NOW (CHAIN 1).
Chain 1:
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NA
I’m using this as a step-wise experiment and only testing the mock feeds with the optimal locus combination:
# make a list of just the four loci
four_locs <- c("mifish", "nsCOIFo", "fishminiA", "cep")
Now loop over those four for the feeds and mock feed pools
# lapply over the four loci for the mock equal pool replicates
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "MEP", n_replicates = 9)
Now loop over those four for the feeds and mock feed pools
# lapply over the replicates for the mock feed pool and the top four loci
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "MFP", n_replicates = 9)
# lapply over the four loci for filler2 and 0% fishmeal replicates
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "F2_0", n_replicates = 6)
# lapply over the four loci for filler1 and 0% fishmeal replicates
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "F1_0", n_replicates = 6)
# lapply over the four loci for 100% fishmeal replicates
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "F1_100", n_replicates = 6)
# lapply over the four loci for filler1 and 2% fishmeal replicates
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "F1_2", n_replicates = 6)
# lapply over the four loci for filler1 and 25% fishmeal replicates
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "F1_25", n_replicates = 6)
# Workaround for the F1_10% feeds to avoid including the 100% fishmeal replicates as well.
feed10per_feature_df <- features_w_taxonomy %>%
filter(str_detect(sample, "F1_10")) %>%
filter(!str_detect(sample, "F1_100"))
# lapply over the four loci for filler 1 and 10% fishmeal reps
lapply(four_locs, sodm.by.locus, feature_table = feed10per_feature_df, site = "F1_10", n_replicates = 6)
# lapply over the four loci for filler 2 and 25% fishmeal reps
lapply(four_locs, sodm.by.locus, feature_table = features_w_taxonomy, site = "F2_25", n_replicates = 6)
Overall, the cleanest signal is undoubtedly from the vouchered reference pool. I’m going to need to filter according to the locus-ASV and sample, so I can’t just combine the whole set of .csv files at this stage.
I’ll organize and filter the output files in the next R notebook.